Download No Slide Title

ESSENTIALS OF GLYCOBIOLOGY
LECTURE 16
NUCLEAR, CYTOPLASMIC, AND MITOCHONDRIAL
GLYCOSYLATION
Hud Freeze
Traditional Glycobiology Studies
ER- and Golgi-based Glycosylation
MAJOR FORMS OF CYTOPLASMIC GLYCOSYALTION
Animals: Hyaluronan, Glycogen
Plants: Cellulose
Insects Yeast & Fungi: Chitin
MITOCHONDRIAL GLYCOSYLATION
FACT OR FICTION?
SUGGESTIVE RESULTS FROM THE EARLY DAYS
•Suggested by radio labeling with sugar precursors
•Lectin binding studies
SOME NAGGING PROBLEMS
•Contamination by other cellular components, esp ER
•How to get glycosyltransferases into mitochondria?
•How do substrates enter?
CLOSE FRIENDS ??
MITOCH
ER
QuickTime™ and a
Video decompressor
are needed to see this picture.
?
GLYCOSYLATION IN MITOCHONDRIA?
Metabolic labeling with 35S +/- deglycosylation
3H-Mannose
labeling
Specific mitochondrial protein complexes label with 3H-Man
F1-ATPase
(Complex-V)
NADH-Ubiquinone-(Complex I)
The glycoprotein binds to
The lectin ConA
Nuclear and Cytoplasmic Glycosylation
In Favor
Opposed
• Many papers
• Orientation of
Transferases- Type II
Membrane Proteins
• Highly regarded journals
• Many
• Lectins
 Nuclear Localization
Signals
• Composition
 No site mapping
• Metabolic Labeling
•No structural analysis of
glycan
•Enzyme digestions
•Purity
CYTOSOLIC GLYCOSYLATION
CYTOSOLIC GLYCOSYLATION
Glycogenin is a Self Priming Glucosyl transferase
3
GN
GN
2
Tyr-194
Tyr-194
GS
UDP
1
UDP-Glc
GN
Tyr-194
GS
Phosphorylase+
Debranching Enzyme
UDP-Glc
UDP
4
GS
GS
Tyr-194
GN
Tyr-194
GN
5
Branching Enz+GS
Intersubunit Priming
Tyr-194
Tyr-194
Intrasubunit Priming
Tyr-194
Tyr-194
CYTOSOLIC GLYCOSYLATION
Stages of Dictyostelium
Development
Skp1 glycosylation pathway in Dictyostelium
Most complex form of cytoplasmic glycosylation
Verified Complex Cytoplasmic Glycosylation
• Only demonstrated for a single protein, SkpI, in
Dictyostelium
• SkpI involved in ubiquitination of cell cycle
proteins
• Attached O-linked chain at Pro-143 (not
conserved) which is first hydroxylated to HyPro
• Gala-6GalaFuca1-2Galb1-3-GlcNAc-HyPro
• GlcNAc and Fuc Transferase purified and shown
to have very low Km for sugar nucleotides
• First Gal and Fuc are added by single
transferase
• Mammalian homologs may not have this
modification
CYTOPLASMIC GLYCOSYLATION
O-Mannose
•UDP-Glc Transfers Glc1-P to Mannose on a cystosolic
protein
•62kD protein is Phosphoglucomutase (Glc-1-PGlc-6-P)
•Transferase and phosphodiesterase found on many
cells
•Membrane association regulated by modification with
Glc-1-P
•Structure of underlying oligosaccharide not known
CYTOPLASMIC GLYCOSYLATION
CYTOSOLIC GLYCOSYLATION
PROTEOGLYCANS MADE IN THE NUCLEUS
NO
DELIVERY TO NUCLEUS?
MAYBE
• GAG SUBFRACTION IS HIGHLY ENRICHED IN ISOLATED NUCLEI
• SMALL FRACTION OF FGF FROM LYSOSOME MAY BE
TRANSPORTED TO THE NUCLEUS
MECHANISM?
CYTOSOLIC GLYCOSYLATION
Ubiquitination of a New Form of a-Synuclein by Parkin from
Human Brain: Implications for Parkinson's Disease
SCIENCE, July 13, 2001
aSp22 is an O-linked glycosylated isoform of a-synuclein.
Failure of parkin to degrade it could cause some forms of PD
Accumulation of an O-glycosylated form of a-synuclein
In autosomal recessive forms of Parkinson’s disease?
CYTOSOL
E3-Ub-ligase
co-incubation with O-glycosidase and sialidase A shifted the 22-kD species to
a 16-kD position (31), where it now co-migrated with the unmodified S
monomer, Sp16, from crude brain extracts (Fig. 5C). We conclude from our
data obtained by mass spectrometry analysis (21) and these enzymatic
digestions that Sp22 is a posttranslationally modified form of human Sp16
containing O-linked sugars.
a3
b3
a-Thr/Ser
31.
For enzymatic digestion of HP2A precipitates, N-glycosidase, sialidase A, endo-Oglycosidase and protein phosphatase-1 (Sigma) were used as per manufacturers'
instructions.
21. The HP2A-specific 22-kD protein yielded tryptic peptides
corresponding to aa 13-21, 44-58, 46-58, 59-80, 61-80, 81-96, and 81-97
of human Sp16 (GenBank accession # L08850), each ending with a
lysine, as expected.
DIGESTION OF O-LINKED GalNAc sugar chain-- 2 easy steps
a3
1.
a-Thr/Ser
b3
SIALIDASE
b3
a-Thr/Ser
2.
b3
O-GLYCOSIDASE
ARE THEY CORRECT OR NOT?
WHAT’S YOUR OPINION?
Summary
•O-GlcNAc is only one form of cytoplasmic glycosylation
• Hyaluronan, glycogen, cellulose and chitin are examples
• Other forms of Cytoplasmic glycosylation exist
Transferases and genes are characterized
• The roles of specific sugar chains in not known in most cases
• Lack of structural information is often a key deficiency
• Mitochondria may acquire N-glycosylated proteins from ER
• Other forms may exist, but proof requires structural analysis